The oleate desaturase acid enzyme (OLD) is involved in the enzymatic conversion of oleic acid to linoleic acid. The microsomal OLD have been cloned and characterized using the marker technology by T-DNA (T-DNA tagging, Okuley, et al. (1994) Plant Cell 6:147-158). Nucleotide sequences of higher plants encoding microsomal OLD have been described in document WO94/11516 PCT of Lightner et al.
Sunflower is generally cultivated to obtain oils that contain saturated fatty acids (palmitic and stearic) and unsaturated fatty acids (oleic and linoleic). The stearic acid content is always lower than 10% (Guston et al. (1986) The lipid handbook, Chapman and Hall Great Britain), usually between 3 and 7%. In relation to the content of unsaturated fatty acids there are two types of sunflower seeds: the normal sunflower, which has a linoleic acid content between 50% to 70% (Knowles (1988) Recent advances in oil crops breeding, AOCS Proceedings) and sunflower with high oleic content, which has a linoleic acid content of 2% to 10% and oleic acid content from 75% to 90% (Soldatov (1976) Chemical mutagenesis in sunflower breeding, Proceedings of the 7th International Sunflower Conference, 352-357). There is also a sunflower line that has a high content of palmitic acid between 22 and 40% (Ivanov et al. (1988) Sunflower Breeding for High Palmitic Acid Content in the Oil, Proceedings of the 12th International Sunflower Conference, Vol II, 453-465) and another line of sunflower with low content of saturated fatty acids (less than 6%) (EP-A-0496504).
In order to respond to the need for vegetable oils of interest to both the industry and to human food consumption, efforts are made in improving varieties of oilseeds focused on modifying the fatty acid composition of seeds, for example, by means of conventional breeding programs, mutagenesis or transgenesis.
Mutations are typically induced with extremely high doses of radiation or chemical mutagens (Gaul (1964) Radiation Botany 4:155-232). High doses exceed the lethal dose 50% (LD50), and generally lethal doses of 90% (LD90) maximize the percentage of possible mutations.
Mutagenesis conducted by Soldatov in 1976 in a population of sunflower allowed obtaining the population of so-called Pervenets mutants. The average content of oleic fatty acid (18:1) of the seeds of this cultivar is higher than 65%, the individual content is between 60 and 80% while in normal varieties (low oleic, LO) this content is approximately 20%. The Pervenets population was distributed worldwide and used in many breeding programs in order to convert certain genotypes with low content of 18:1 into genotypes with a high content of 18:1 in their seed.
The accumulation of 18:1 in seeds is dependent on two enzymatic reactions: the desaturation of 18:0 (stearic acid) to 18:1 and the subsequent desaturation of 18:1 to 18:2 (linoleic acid). The oleate desaturase enzyme (OLD) catalyzes the desaturation of 18:1 to 18:2 (Ohlrogge and Browse (1995) The Plant Cell, 7:957-970, Somerville and Browse (1996) Trends Cell Biol 6:148-153; Schwartzbeck (2001) Phytochemistry, 57:643-652).
Sunflower oil is naturally rich in 18:2 (55-70%) and consequently poor in 18:1 (20-25%). Traditional varieties are classified as low oleic (LO). As there exists a high interest among consumers of eating more healthy oils with high oleic acid content, there is also an interest in developing high oleic sunflower plants, which preferably also lead to similar crop yields as the conventional sunflower varieties.
Studies carried out by Garces et al. in 1989 and 1991 (Garces et al. (1989) Phytochemistry 28:2597-2600; Garces and Mancha (1991) Phytochemistry 30:2127-2130) showed that the high oleic phenotype (high oleic, HO) is associated with a marked decrease in the activity of the OLD enzyme, which catalyzes the desaturation of 18:1 to 18:2 in HO seeds during critical stages of the synthesis of the lipidic reserves, which explains the accumulation of 18:1.
The Pervenets mutation has been shown to be associated with gene duplications within the OLD gene, leading to gene silencing. This decrease in transcription of OLD explains the decrease in the amount of enzyme and therefore the low OLD activity demonstrated (Hongtrakul et al. (1998) Crop Sci 38:1245-1249) consistently to the 18:1 accumulation in sunflower seeds. This finding led to the development of molecular markers characteristic of the mutation that can be used in breeding programs to facilitate the selection of HO genotypes (WO2005/106022; Lacombe et al. (2001) Life Sci 324:839-845).
Traditional sunflower oil with high content of 18:2 is considered a healthy vegetable oil that has a proper taste, and it has been considered first class quality oil in the world market due to its high percentage of polyunsaturated fatty acids. It is used as a salad oil, cooking oil, or for production of margarine.
By modifying the fatty acid profile of sunflower oil, new oil can be developed having higher oxidative stability compared to conventional oil. This oil should at least contain a level of 18:1 of 55% to 65% in relation to the total fatty acids. The benefit of this oil is its high oxidative stability after the extraction process and the stability of the flavor of fried products. Sunflower oil with a high concentration of 18:1 does not need to be hydrogenated to improve stability and does not form trans fatty acids.